SAT-652 - Phosphoenolpyruvate Carboxykinase Enhances Thermogenesis in Human Brown Adipocytes

Obesity is a global health pandemic, responsible for millions of deaths annually worldwide. Brown adipose tissue (BAT) is a promising target against obesity and metabolic disease because it increases energy expenditure through non-shivering thermogenesis. This process is under sympathetic stimulation and mediated primarily by mitochondrial uncoupling protein-1 (UCP1), dissipating the mitochondrial proton gradient to generate heat. We previously showed glucocorticoids acutely increased human brown adipocyte thermogenesis, but decreased activity in murine adipocytes. Bulk RNAseq of human and murine brown/ beige adipocytes incubated with cortisol/ placebo for 24h revealed expression of PCK1, encoding phosphoenolpyruvate carboxykinase (PCK), was increased ~2.5-fold in human, but reduced by >50% in murine adipocytes. Both cytosolic PCK1 and mitochondrial PCK2 are key enzymes in glyceroneogenesis and contribute to the replenishment of intracellular triglyceride stores. We hypothesized that PCK plays an important role in human BAT function by linking glucose metabolism to lipid handling.
To identify pathways of glucose utilisation, primary human brown adipocytes were incubated with unlabelled or 13C-glucose. For respirometry/ gene expression studies, siRNA-knockdown of either PCK1, PCK2, PCK1+PCK2 and non-targeted controls was undertaken in human brown adipocytes.
In human brown adipose tissue, both PCK1 and PCK2 were highly expressed, althouh PCK2 was more highly expressed in human brown adipocytes. PCK1 mRNA levels were increased following noradrenaline stimulation, and its expression closely correlated with UCP1 in human BAT. 13C-glucose tracing in human brown adipocytes revealed substantial 3-phosphoglycerate, dihydroxyacetone phosphate, and glycerol-3-phosphate synthesis, which was increased further following noradrenaline stimulation, consistent with glyceroneogenesis during brown adipocyte thermogenesis. Interestingly, siRNA-knockdown of PCK2 (but not PCK1) reduced mitochondrial respiration, which was decreased further by combined PCK1/ PCK2 knockdown.
These findings suggest a previously underappreciated role for PCK in coordinating glucose-derived carbon flux with thermogenic function in human BAT.

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